Although it is generally accepted that interstitial cells of the testis are a primary source of androgenic steroid(s) (testosterone or its precursors) and that these hormones influence activities in the seminiferous tubules, the discrete sites of origin, transport, and action of specific steroid hormones in the testis have not been identified. We propose to use the unlabeled antibody, peroxidase-antiperoxidase complex technique of Sternberger, et al., as modified by Moriarty and Halmi, to identify the location of testosterone in the testis of normal adult and hypophysectomized rats, and in interstitial and Sertoli cells studied in vitro. Our preliminary studies suggested that anti-testosterone (1:1000 to 1:4000) bound to residual bodies and lipid inclusions in the cytoplasm of Sertoli cells. When the antitestosterone was diluted, there was a decrease in the intensity of the stain. No staining was observed when normal rabbit serum was substituted for anti-testosterone. When goat anti-rabbit serum or PAP was omitted from the procedure, staining did not take place. Furthermore, when anti-testosterone was first absorbed with testosterone (1-10 ng/ml), staining did not occur. Tissue will be fixed with different combinations of solutions to determine that method which best immobilizes and preserves the immunoreactivity of steroids in tissue. To determine the specificity of the reaction, anti-testosterone will be absorbed with testosterone, 5alpha-dihydrotestosterone, androstenedione, and 17alpha-epitestosterone. Staining will be quantitated by counting PAP complexes on electron micrographs by means of a stereoscopic microscope equipped with an ocular grid. If the locations of testosterone (and other steroids) in the testis can be identified at the ultrastructural level, this morphological evidence can be correlated with information gained from biochemical studies to further clarify steroid biosynthesis in the testis.